Rapid chemical reaction techniques play an important role in unraveling the mechanism of reactions mediated by soluble proteins, including enzymes involved in the regulation of intracellular processes and the biosynthesis of proteins and nucleic acids. Regulatory proteins change conformation rapidly and must, therefore, be studied in the microsecond-to-millisecond time region. Similar techniques for investigating reactions mediated by membrane-bound neurotransmitter receptors were not available, and the mechanisms of the reactions are poorly understood. These proteins must be studied in a membrane-bound form in cells or vesicles, over a wide range of reactant concentrations, and in the microsecond-to-millisecond time region. Four rapid-mixing techniques for making kinetic measurements in the microsecond and millisecond time regions have now been developed for investigating neurotransmitter receptors in the membranes of neurons and muscle cells, thus extending chemical kinetic approaches to membrane-bound proteins and intercellular processes. Neurotransmitter receptors regulate transmission of signals between neurons (approximately 10(12) in the human nervous system), thereby allowing perception of stimuli, integration and storage of information, and reaction to the environment. Six structurally related neurotransmitter receptors, and many isoforms, have been identified by use of recombinant DNA technology. Modern electrophysiological techniques show that these different proteins, upon binding a specific chemical signal (neurotransmitter), transiently open transmembrane channels, which are characterized by their ion selectivity, conductance, and lifetime. To be able to account for the receptor-mediated voltage changes that trigger signal transmission between cells, we still need to know the concentration of open receptor-channels. This concentration changes with time and is affected by the concentration of neurotransmitter. Rapid reaction techniques are particularly suitable for determining the relationship between neurotransmitter concentration and the time-dependent concentration of the open receptor-channels. The four rapid reaction techniques adapted or developed for studying receptor mechanisms are quench- and stopped-flow, adapted for use with vesicles, and cell-flow and laser-pulse photolysis for use with single cells. The approach was initiated when it was found that the neurotransmitter receptors desensitize (become transiently inactive) faster, by almost two orders of magnitude, than was believed. Before fast reaction techniques were used, the chemical properties of only desensitized forms were investigated, although this was not recognize. So far, the chemical mechanism(s) of the excitatory (cation-specific) acetylcholine receptor in membrane vesicles, electroplax cells, and single clonal cells, and the inhibitory (anion-specific) gamma-aminobutyric acid (GABA) receptor in primary cerebral cortical cells have been investigated with the new techniques. A minimum mechanism and its constants for the acetylcholine receptor from the Electrophorus electricus electroplax have been determined. Together, they account for the concentration of open channels and desensitization rates over a 5000-fold range of acetylcholine concentration. The mechanism appears also to account for the muscle and neuronal acetylcholine receptor, and for the structurally related GABA receptor. When comparison has been possible, the results obtained by chemical kinetic techniques also accounted for results obtained by the single-channel current recording technique. Measurements of the conductance and lifetime of receptor-formed transmembrane channels enhanced our understanding of (1) differences between receptor isoforms in different cells, (2) changes in receptor mechanism during development, or produced by protein engineering, (3) changes in receptor mechanism relevant to diseases of the nervous system, and (4) the effects of therapeutic drugs and abused compounds. The new techniques can be used in all these areas to measure the effect of neurotransmitter concentration on channel opening and on receptor desensitization and resensitization, and to determine and combine constants of individual steps of the reaction pathway into an overall chemical mechanism. Thus, it is now becoming possible to relate the receptor-mediated reaction, and factors that affect it, to changes in the transmembrane voltage of the cell, and therefore to the transmission of signals between cells of the nervous system.
